Design, synthesis, and cocrystal structure of a nonpeptide Src SH2 domain ligand

Article abstract of DOI:10.1021/jm970402q

The specific association of an SH2 domain with a phosphotyrosine (pTyr)- containing sequence of another protein precipitates a cascade of intracellular molecular interactions (signals) which effect a wide range of intracellular processes. The nonreceptor

Full text of DOI:10.1021/jm970402q

J . Med. Chem. 1997, 40, 3719-3725
3719
Design , Syn th esis, a n d Cocr ysta l Str u ctu r e of a Non p ep tid e Sr c SH2 Dom a in
Liga n d
Mark S. Plummer,* Debra R. Holland, Aurash Shahripour, Elizabeth A. Lunney, J ames H. Fergus,
J ames S. Marks, Patrick McConnell, W. Thomas Mueller, and Tomi K. Sawyer
Departments of Chemistry, Biochemistry, and Biotechnology, Parke-Davis Pharmaceutical Research Division,
Warner-Lambert Company, 2800 Plymouth Road, Ann Arbor, Michigan 48105
Received J une 17, 1997X
The specific association of an SH2 domain with a phosphotyrosine (pTyr)-containing sequence
of another protein precipitates a cascade of intracellular molecular interactions (signals) which
effect a wide range of intracellular processes. The nonreceptor tyrosine kinase Src, which has
been associated with breast cancer and osteoporosis, contains an SH2 domain. Inhibition of
Src SH2-phosphoprotein interactions by small molecules will aid biological proof-of-concept
studies which may lead to the development of novel therapeutic agents. Structure-based design
efforts have focused on reducing the size and charge of Src SH2 ligands while increasing their
ability to penetrate cells and reach the intracellular Src SH2 domain target. In this report we
describe the synthesis, binding affinity, and Src SH2 cocrystal structure of a small, novel,
nonpeptide, urea-containing SH2 domain ligand.
In tr od u ction
two well-defined pockets, the pTyr (pY) pocket and the
hydrophobic binding pocket (pY+3). Using the three-
dimensional X-ray structure of Src SH2 domain com-
plexed to the 11-mer phosphopeptide, Glu-Pro-Gln-pTyr-
Glu-Glu-Ile-Pro-Ile-Tyr-Leu, a structure-based design
approach has been employed to discover small molecule
Src SH2 ligands. The first reported efforts reduced the
11-mer peptide to a potent pentapeptide,17 and then
later to a homologated D-amino acid containing tripep-
tide.18 Previously, we have communicated our design
of a series of D-amino acid containing tripeptide ligands,19
a series of dipeptide ligands,20 and our efforts to modify
the pTyr residue.21,22
Our motivation for reducing the size, charge, and
peptide character of these ligands, while maintaining
the potency, is derived from the fact that SH2 domains
are intracellular targets requiring the ligands to pen-
etrate cell membranes. Ultimately, inhibition of SH2-
phosphoprotein interactions by such small molecules
will aid biological proof-of-concept studies which may
lead to the development of novel therapeutic agents
designed to attenuate Src SH2 specific signal transduc-
tion pathways. In this paper, we describe the synthesis,
binding affinity, and Src SH2 cocrystal structure of a
novel nonpeptide, urea-containing ligand for the Src
SH2 domain.
The pp60c-src (Src) protein is a nonreceptor tyrosine
kinase which is characterized by an N-terminal unique
domain followed by two Src homology domains, SH3 and
SH2, a catalytic kinase (or SH1) domain, and a short
C-terminal regulatory peptide segment. SH2 domains
consist of approximately 100 amino acids and bind to
phosphotyrosine (pTyr)-containing proteins in a se-
quence dependent manner, while SH3 domains bind to
proline-rich sequences in peptides. The association of
an SH2 domain with a pTyr-containing sequence of
another protein is believed to initiate a complex cascade
of intracellular molecular interactions (signals), which
effect a wide range of processes including cell growth,
proliferation, mitogenisis, and transformation.1-3 Phos-
phoproteins which have been shown to bind to the Src
SH2 domain include middle T antigen, PDGF receptor,
EGF receptor, and focal adhesion kinase.4-8 Further-
more, Src has been implicated to play a role in both
breast cancer5 and osteopetrosis,9,10 suggesting that Src
might be an attractive therapeutic target.
We have chosen to focus our drug design efforts on
the Src SH2 domain since its three-dimensional struc-
ture was the first to be determined by X-ray crystal-
lography and since SH2 domains recognize short syn-
thetic pTyr-containing peptides, in which specificity is
conveyed by the residues immediately C-terminal to the
pTyr residue.11,12 For Src the optimal binding sequence
was found to be -pTyr-Glu-Glu-Ile-. Extensive crystal-
lographic studies have provided a detailed molecular
map of both complexed and uncomplexed forms of the
Src SH2 protein.13-15 Since that time, the three-
dimensional structures of numerous SH2 domains have
also been solved in both liganded and apo-protein forms
by X-ray crystallography and NMR techniques.16 Struc-
turally, all SH2 domains studied thus far are comprised
of a central antiparallel â-sheet core flanked on each
side by an R-helix. In the reported Src structures, as
in other SH2 structures, the phosphopeptide ligands
bind perpendicular to the â-sheet core and interact with
Ch em istr y
The known pentapeptide,17 1, was synthesized using
solid-phase methodology based on an Fmoc/tert-butyl
protection strategy.23 The resin for the synthesis of 1
was split prior to the incorporation of the final tyrosine
residue, allowing incorporation of tyrosine mimic, 8, to
yield compound 2. The tyrosine mimic (R)-2-(p-hy-
droxybenzyl) succinic acid, 8, was synthesized by adapt-
ing the known Evans methodology-based synthesis of
the related phenylalanine mimic.24 The synthesis of
tripeptide 3, dipeptide 4, and the protected amine 9,
used in the synthesis of Src SH2 ligands 5 and 6, has
been previously reported.20 The intermediate 9 was
deprotected providing the amine 10 which was em-
ployed, without purification, in conjunction with N-(p-
X Abstract published in Advance ACS Abstracts, October 15, 1997.
S0022-2623(97)00402-0 CCC: $14.00 © 1997 American Chemical Society

3720 J ournal of Medicinal Chemistry, 1997, Vol. 40, No. 23
Plummer et al.
Sch em e 1a
a
(a) 20% piperdine, CH2Cl2, 20 min; toluene, evaporate; (b) ref 25; (c) H2, 5% Pd on C THF/MeOH; (d) THF, di-tert-butyl
diethylphosphoramidite, tetrazole, then t-BuOOH; (e) CF3CO2H:CH2Cl2:H2O (95:4.5:0.5).
Sch em e 2a
ally directly deprotected with 95:4.5:0.5, trifluoroacetic
acid:dichloromethane:water. Purification of the crude
phosphorylated Src SH2 ligand was accomplished by re-
versed phase preparative HPLC providing 5, 6, and 7.
Resu lts a n d Discu ssion
Design Str a tegy. Recently, we have demonstrated
the cellular delivery of a series of dipeptide Src SH2
ligands.26 This was accomplished by replacement of the
phosphate group of pTyr, which is expected to be
dephosphorylated by nonspecific phosphatases inside
cells, by the nonhydrolyzable 4-(phosphonodifluoro-
methyl)phenylalanine (F2Pmp) group.27,28 The F2Pmp
group, which is also dianionic at physiological pH, is
then masked as the (acyloxy)methyl ester to provide
prodrugs which both penetrate cells and are readily re-
converted to the parent Src SH2 ligands inside the cell.
Further development of small effective Src SH2
ligands, which both penetrate cells and provide insight
into the therapeutic potential of blocking SH2 domain-
phosphoprotein interactions, may hinge on discovering
simplified pTyr replacements which ideally would be
uncharged and achiral. To this end we synthesized a
number of Ac-pTyr analogues21,22 and incorporated them
into the pTyr-Glu-Glu-Ile-Glu sequence for convenient
comparison to previously studied phosphate replace-
ments reported by Glaxo researchers.17 The most potent
Ac-Tyr mimic, 8, was obtained by replacing the aceta-
mide group with an R-carboxymethyl group providing
analogue 2 which was equipotent with the parent
compound, 1, Ac-pTyr-Glu-Glu-Ile-Glu (Table 1). Mo-
lecular modeling of 2 suggests that potency is retained
due to the N-terminal carboxy group forming a bifur-
cated hydrogen bond to the side chain of Arg 12 just as
the acetamide group does in the pentapeptide, 1,
complex.17 A similar hydrogen bond was seen in the
X-ray structure of the D-amino acid containing tripeptide
3 complexed to Src SH2.32
a
(a) HCl‚Glu(Ot-Bu)OMe, ClCOCl in toluene, pyridine, CH2Cl2,
0 °C, then 6-amino-1-naphthol; (b) KOH, H2O/MeOH; (c) methyl(3-
cyclohexylpropyl)amine, EDCI, HOBT, N-Me-morpholine; (d)
CH2Cl2, di-tert-butyl diethylphosphoramidite, tetrazole; t-BuOOH;
(e) CF3CO2H:CH2Cl2:H2O (95:4.5:0.5).
hydroxybenzyl) glycine tert-butyl ester, 11, to form urea
13 using the methodology of Nowick (Scheme 1).25
Likewise, amine 10 was employed in conjunction with
4-(benzyloxy)benzylamine 12 to form urea 14. Hydro-
genolysis of the benzyl protecting group in 14 gives the
phenol 15 (Scheme 1). The naphthylurea-containing Src
SH2 ligand, 7, (Scheme 2) was synthesized by first
reacting Glu(OtBu)-OMe in dichloromethane and excess
pyridine with phosgene in toluene to form the isocyan-
ate, which was subsequently coupled with 1-amino-6-
naphthol to give urea 18. The methyl ester of 18 was
hydrolyzed, and the resulting acid, 19, was coupled with
methyl (3-cyclohexylpropyl)amine to provide urea 20.
The phenol functionality in 13, 15, and 20 was phos-
phorylated by employing di-tert-butyl diethylphosphor-
amidite and tetrazole (Schemes 1 and 2). The phosphi-
tylated phenol was directly oxidized in situ with tert-
butyl hydroperoxide, providing the protected phosphates
16, 17, and 21, which could be isolated but were typic-
Rather than incorporating the (R)-(p-hydroxybenzyl)
succinic acid, 8, Tyr mimic used in peptide 2 into our
dipeptide series of compounds, simplification by remov-
ing the chiral center appeared advantageous since the
achiral N-(p-hydroxybenzyl)glycine analogue was readily

Synthesis of a Nonpeptide Src SH2 Domain Ligand
J ournal of Medicinal Chemistry, 1997, Vol. 40, No. 23 3721
Ta ble 1
synthesized.30,31 The protected achiral Tyr mimetic 11
was substituted for the Ac-Tyr group in dipeptide 4 (IC50
) 0.8 µM) to provide 5 (IC50 ) 7.0 µM) resulting in about
a 10-fold loss of potency (Table 1). Initially, this drop
in potency was hypothetically attributed to the achiral
sp2-hybridized urea linkage causing a ligand conforma-
tion unfavorable for optimal binding. Structurally, loss
of the sp3 hybridized chiral center could cause the phos-
phate group to be poorly positioned in the pY pocket.
Alternatively, the urea linkage could cause the N-
terminal carboxy group to be outside hydrogen bonding
distance with the Arg 12 side chain. Modeling predicted
that the carboxy group of 5 was capable of forming a
bifurcated hydrogen bond with the Arg 12 side chain
while the phosphophenyl group occupied the pY pocket.
However, other interactions might be compromised.
To directly evaluate the role of the carboxy group in
ligand binding, the Tyr mimic 11 was further simplified
to p-hydroxybenzylamine providing the nonpeptide Src
SH2 ligand 6 (Table 1). Importantly, 6 (IC50 ) 12 µM),
which lacks the carboxymethyl group of 5, is only about
2-fold less potent than 5. This suggests that the loss of
binding affinity of 5 and 6 relative to dipeptide 4 is likely
attributable not only to a conformational effect induced
by the achiral urea on the position of the phenyl
phosphate group but also to a diminished interaction
of the carboxy group of 5 with Arg 12san interaction
not possible with 6 which lacks the carboxy group.
To explore the effect of restricting the conformational
freedom of the p-hydroxybenzylamine derived portion
of 6, 1-amino-6-naphthol was employed next as a Tyr
mimic (Table 1). This modification, in conjunction with
the urea linkage, restricts rotational freedom of ligand
7 from the glutamic acid NH through to the phosphate
group (as compared to the Ac-pTyr dipeptide analogue
4). Nonpeptide 7 (IC50 ) 13 µM) is equipotent with 6,
demonstrating that the increased size and decreased
rotational freedom imposed by the naphthol-based Tyr
mimic is accommodated by the phosphate binding
pocket of Src SH2. These results also suggest that this
pocket is somewhat adaptable. The approximate 10-
fold difference in binding affinity between 4 and non-
peptides 5, 6, and 7, which contain a diverse set of Ac-
Tyr mimics, may be partially attributed to diminished
hydrogen-bonding interactions of these Tyr mimics,
relative to Ac-Tyr, with the Arg 12 side chain. Even

3722 J ournal of Medicinal Chemistry, 1997, Vol. 40, No. 23
Plummer et al.
F igu r e 1. Divergent stereoview of X-ray structures for binding sites of Src SH2 bound to urea 5 (colored by atom type: C,
yellow; O, red; N, blue; P, white) and tripeptide 3 (colored cyan). The structural water of the tripeptide complex is shown as a
cyan sphere and is labeled as “W”. The initial 2.4 Å residual map for urea 5 (colored magenta) is contoured at 3σ and superimposed
over the final model of the ligand.
F igu r e 2. Divergent stereoview of the refined 2.4 Å 2Fo - Fc electron density map showing bound ligand (thick bonds) and the
surrounding protein residues (thin bonds). The map is contoured at 1σ and 2σ.
with a somewhat adaptable pTyr binding pocket, poor
positioning of the phosphate in the binding pocket could
also play a role in the diminished binding affinity.
Cr ysta l Str u ctu r e of Ur ea 5. To gain an under-
standing of the nature of the interactions between the
SH2 domain and the nonpeptide ligands 5 and 6,
cocrystallization experiments were conducted. A crystal
structure of 5 complexed with Src SH2 was determined.
An initial Fo - Fc residual map, phased on the molecular
replacement solution without ligand, shows the urea
compound 5 in good detail (Figure 1). The overall
structure of the SH2 domain in the complex is very
similar to that previously described,13,14,17 and the
quality for the final 2.4 Å 2Fo - Fc map is excellent, as
it fully describes both the protein and ligand (Figure
2). Src SH2 residues 107-112 are disordered according
to the electron density map and are therefore not
included in the final model.
The X-ray results do reveal, however, a unique ligand
binding mode which is different than that previously
reported for Src SH2 peptide ligands, including the
dipeptide 432 and the tripeptide 3.32 Although ligand 5
binds with the phosphate group in the pY pocket and
the cyclohexyl group in the hydrophobic pY + 3 pocket,
as expected, the conformation of the ligand is quite
different than initial predictions (Figure 1). In particu-
lar, the orientation of the phenyl phosphate ring in the
pY pocket is nearly perpendicular to that exhibited by
tyrosine-containing peptide ligands, although the phos-
phate group is centered within 1 Å of that observed for
the peptide ligands. Also, as if to avoid a steric clash
with the phenyl phosphate ring, the guanadinium of Arg
12 moves away from the pY pocket by several angstroms
and, as a result, is not able to form the expected hy-
drogen bond with the ligand N-terminal carboxymethyl
group, leaving it completely exposed to solvent. The side
chain of Lys 60 also moves to accommodate the differ-
ence in phosphophenyl binding.
Most surprising is the existence of a cis-amide linkage
between the pY + 1 Glu and the C-terminal methyl (3-
cyclohexyl propyl) amine group with respect to dipeptide
432 which has this group trans-oriented. As a result of
this unexpected configuration, the structural water
molecule (shown as sphere in Figure 1), which usually
mediates a hydrogen bond between the Lys 60 backbone
and the pY + 1 ligand carbonyl of more peptidic Src SH2
ligands,13,14,17 is displaced by the cis-amide carbonyl
oxygen to provide a new direct protein-ligand contact.
The hydrogen bond between His 58 carbonyl and the
pY + 1 Glu NH observed in all other reported peptide
complexes is preserved in the ureido complex, as is the
solvent-exposed environment of the pY + 1 Glu side
chain. The C-terminal cyclohexyl ring of 5, however,
does not bind as deeply into the pY + 3 pocket as does
the preferred Ile residue of the high affinity pY-E-E-
I-E sequence, but more deeply than the cyclohexyl group
of the tripeptide 3.
Figure 3 shows the hydrogen bond and van der Waals
contacts made by the urea ligand with the Src SH2
domain. In spite of the unique binding mode for the
phenyl phosphate group within the pY pocket, the
phosphate moiety assumes the same position, but has
a different relative orientation, when compared with the
phosphate of other Src SH2 ligand complexes (Figure
1). The same pY pocket side chains are involved in the
hydrogen-bonding scheme of the phosphate with the
exception of Cys 42 and Lys 60, each of which makes
contact with a phosphate oxygen in the urea complex,
but not in other peptide complexes. However, Arg 12
does not make a hydrogen bond with the phosphate of
the urea ligand while Arg 12 does in other crystal
complexes reported.

Synthesis of a Nonpeptide Src SH2 Domain Ligand
J ournal of Medicinal Chemistry, 1997, Vol. 40, No. 23 3723
SH2 fusion protein was performed in 20 mM Tris (pH 7.5),
150 mM NaCl, 5 mM EDTA, and 0.1% NP-40. Assay additions
to a Millipore filter plate (0.45 mM PVDF) resulted in v-Src
SH2 fusion protein-glutathione sepharose bead complex, 2.8
nm [125I]phosphopeptide and 2% DMSO + test compound at
different concentrations. Binding was performed at room
temperature for 20 min while continuously inverting the plate.
Unbound [125I]phosphopeptide was separated from SH2-bound
radiolabeled peptide by vacuum filtration and washing two
times with 100 µL per well of assay buffer. Results are
expressed as IC50 values and are averages of at least two
duplicate determinations.
X-r a y Meth od s. As described,29 our v-Src SH2 domain was
expressed in E. coli as a 112-amino acid protein and stored as
an ammonium sulfate precipitate slurry (70%). A sample of
the slurry was dialyzed against 100 mM NaCl, 10 mM Tris‚
HCl, pH 7, and concentrated to 30 mg/mL. The complex of
Src SH2 with the urea compound 5 was crystallized at room
temperature by vapor diffusion in a hanging drop consisting
of mother liquor, protein, and 4 mg/mL ligand in a 4:4:2 ratio,
with the mother liquor being 20% PEG 6000 and 100 mM
sodium cacodylate, pH 6. Hexagonal rods (0.35 mm × 0.1 mm
× 0.1 mm) appeared within a week. X-ray diffraction data
were collected on a MarResearch image plate, mounted on a
Rigaku 200B rotating anode generator (operating at 40 kV and
120 mA) and a graphite monochromator. The data were
processed using the XDS package33 to determine the space
group as P61 with one molecule in the unit cell and cell
dimensions a ) b ) 74.4 Å, c ) 38.8 Å (VM ) 2.2 Å3/Da). Using
a detector distance of 100 mm and 1200 s/frame exposures,
95.5% of data 2.4-20.0 Å was collected (4692 unique reflec-
tions) with an R-merge of 10.5% and a multiplicity of four.
The crystal structure of the complex was determined by
AmoRe molecular replacement methods34 using 3.5-10 Å data
and the Src SH2 molecule in complex with the 11-mer
phosphopeptide13 as a search model. The search model did
not contain the phosphopeptide or the water molecules. The
unambiguous molecular replacement solution gave a correla-
tion coefficient of 60.5% and an R factor of 36.2%. An initial
2.4 Å Fo - Fc electron density map, phased on the molecular
replacement solution, confirmed the correct solution had been
found since density in the binding site closely resembled the
urea compound 5. The structure was refined at 2.4 Å resolu-
tion using conventional refinement (positional and indepen-
dent B factor) in XPLOR35 and manual model building was
performed in QUANTA36 on a Silicon Graphics Indigo2 work-
station. Waters were added to the model as the quality of the
maps allowed. The final R factor was 20% with 67 water
molecules. Overall deviations from ideal geometry for bonds,
angles, planes, and impropers were 0.014 Å, 2.9°, 2.9°, and
28.6°, respectively. The coordinates for the complex have been
deposited in Brookhaven DataBank.
Ch em istr y. Gen er a l. 1H NMR spectra were recorded on
either a Varian XL300 or Unity 400 spectrometers in DMSO-
d6 (DMSO) unless otherwise indicated. 31P NMR spectra were
obtained on a Varian Unity 400 spectrometer using a 5 mm
Nalorac Quad probe. Spectra were obtained at 293 K, and
phosphorus chemical shifts are referenced to external H3PO4
in CDCl3 at 0.0 ppm. Chemical ionization mass spectra were
recorded on a Fisons (VG Biotech) Trio-2A using 1% ammonia
in methane as the reagent gas. Electrospray and APCI mass
spectra were obtained on a single quadrapole instrument (VG
Fisons Trio 2000). Flash silica gel chromatography was
performed using Keiselgel 230-400 mesh. Analytical HPLC
chromatograms were obtained using a C18 analytical column
(Vydac, 4.6 × 250 mm), eluting with a linear gradient of
0-66% acetonitrile containing 0.1% TFA and water containing
0.1% TFA over 22 min unless otherwise indicated, and the
peaks were detected at 214 nm. Thin-layer chromatography
employed EM Science silica gel 60 F254 glass backed plates.
Tetrahydrofuran (THF) was dried over sodium-benzophenone
ketyl and distilled. All other commercially available solvents
and reagents were used without further purification. The
synthesis of compounds 1, 3, and 4 have been previously
described.17,19,20
F igu r e 3. LIGPLOT38 representation of hydrogen bonds
(dashed lines) and van der Waals contacts (spiked partial
circles) made by urea 5 (thick bonds) and Src SH2 domain (thin
bonds). The gray color scale: phosphorus, white; nitrogen,
light; oxygen, medium; carbon, dark.
Con clu sion s
On the basis of the structural information provided
by the X-ray analysis of the complex, the 10-fold
difference is potency between ligands 4 and 5 can
probably be attributed to several factors: (1) the lack
of interaction between the SH2 Arg 12 side chain and
both the N-terminal carboxy moiety and the phosphate
group of the ligand 5, (2) the difference in the phosphate
binding pattern in the pY pocket, and (3) the displace-
ment of the structural water of the peptide complexes
with the cis-amide carbonyl. In spite of the decrease
in potency, we have successfully designed a series of
nonpeptide Src SH2 ligands which contain simplified
Tyr mimics. The design was based on modeling of
dipeptide 4 in the context of the tripeptide 3 crystal
complex. These mimics may aid in the discovery of
small effective Src SH2 ligands, which penetrate cells
and block Src SH2-phosphoprotein interactions. The
crystal structure of 5 complexed with Src SH2 domain
reveals some important and unique binding interactions
not observed in the complex with tripeptide 3. Both the
orthogonally orientated phenyl phosphate group in the
pY pocket and the unique conformation of the solvent-
replacing cis-amide backbone suggest that a potent
nonpeptide Src SH2 ligand need not be strictly modeled
after the conventional peptide binding mode. An un-
derstanding of these differences may enable us to design
more potent and further simplified Src SH2 ligands.
Exp er im en ta l Section
In h ib it ion of [125I]P h osp h op ep t id e Bin d in g t o Im -
m obilized Sr c SH2. The binding affinities of compounds
tothe v-Src SH2 domain was determined using a competitive
radiolabeled phosphonopeptide displacement assay. Specifi-
cally, binding of 125I-labeled Glu-Pro-Gln-F2Pmp-Glu-Glu-Ile-
Pro-Ile-Tyr-Leu to a glutathione-S-transferase(GST)-v-Src
Syn th esis of P ep tid es 1, 2, a n d 3. The nonphosphory-
lated peptides were prepared by standard solid-phase synthetic

3724 J ournal of Medicinal Chemistry, 1997, Vol. 40, No. 23
Plummer et al.
1
methodology employing a manual shaker. The peptides were
assembled using (4-(hydroxymethyl)phenoxy)methyl (HMP)
polystyrene resin to give the C-terminal carboxylic acid
derivatives or 4-[(2′,4′-dimethoxyphenyl)-Fmoc-aminomethyl]-
phenoxy (Rink) resin for the C-terminal carboxamide deriva-
tive (giving compound 3)19 and were synthesized using Fmoc/
tert-butyl strategy with 1-hydroxybenzotriazole/diisopropyl-
carbodiimide mediated coupling in N-methylpyrrolidinone. The
resin was split after coupling the first four protected amino
acids (Glu-Glu-Ile-Glu-resin) to allow incorporation of Ac-Tyr
to give known 117 or the Tyr mimic 8. The tyrosine residue
(or mimic) was incorporated with the phenolic residue unpro-
tected, allowing phosphorylation on the resin. Phosphorylation
was accomplished using di-tert-butyl N,N-diethylphosphor-
amidate (50 equiv) and tetrazole (150 equiv) in anhydrous
tetrahydrofuran followed by oxidation with tert-butyl hydro-
peroxide according to a described procedure.37 The peptide was
cleaved from the resin using TFA/water/thioanisole (20:1:1,
v/v). The phosphopeptide 2 was isolated by preparative
reversed phase HPLC. HPLC 100%, tR ) 11.4 min, C18,
eluting with a gradient of 0-66% acetonitrile containing 0.1%
TFA, and water containing 0.1% TFA over 22 min. Electro-
spray mass spectrum (50/50 acetonitrile/water + 0.1% am-
monium hydroxide) m/z 804 (M - H). Anal. (C32H54N4O18P1‚
(0.50)H2O‚(0.91)TFA‚(0.46)CH3CN) C, H, N.
4-[3-(Ca r boxym eth yl)-3-[4-(p h osp h on ooxy)ben zyl]u r e-
id o]-4-[(3-cycloh exylp r op yl)m et h ylca r b a m oyl]b u t yr ic
Acid (5). (Step 1) To Glu(OtBu)-N(Me)-3-cyclohexylpropyl, 10
(360 mg, 1.0 mmol), synthesized as previously described,20 ice,
dichloromethane (20 mL), and saturated sodium bicarbonate
(20 mL), in a separatory funnel was added phosgene in toluene
(7.5 equiv). The mixture was shaken for 5 min, and then the
organic layer was separated, dried over magnesium sulfate,
and concentrated to give the isocyanate as a colorless oil. The
isocyanate was treated in dichloromethane (20 mL) with (4-
hydroxybenzyl)glycine tert-butyl ester, 11 (250 mg, 1.0 mmol),
and triethylamine (0.15 mL, 1.0 mmol) for 1 h. Ethyl acetate
was added, and the reaction mixture was washed with 10%
sulfuric acid, then water, and then brine. After the mixture
was dried over magnesium sulfate, the solvent was removed
under reduced pressure to give the urea 13 as a colorless oil
(500 mg, 83%): 1H NMR (400 MHz) 0.92 (m, 2H), 1.20-2.20
(m, 5H), 1.40 (s, 18H), 1.50-1.70 (m, 10H), 2.22 (m, 2H), 2.90,
3.05 (s, 3H, rotational isomers), 3.10-3.42 (m, 2H), 3.83 (m,
2H), 4.40 (m, 2H), 4.80 (m, 1H), 5.57 (dd, 1H), 6.73 (d, 2H),
7.05 (d, 2H); APCI MS (1:4, methanol/acetonitrile) m/z calcd
603.7, found 604.5 [M + H]+.
genolysis of the benzylurea. The H NMR spectra indicated a
mixture of the starting material 14 and the phenol 15 were
present.
(Step 3) The phenolic hydroxyl group in 15 was phosphity-
lated, oxidized, deprotected, and purified in a manner as
previously described.20 Preparative reversed phase HPLC of
150 mg of crude 6 gave pure product (42 mg) as a colorless
solid after lyophilization: 1H NMR (400 MHz) 1.10 (m, 2H),
1.03-1.26 (m, 5H), 1.40-1.85 (m, 10H), 2.22 (m, 2H), 2.80-
3.01 (s, 3H, rotational isomers), 3.10-3.50 (m, 2H), 4.17 (m,
2H), 4.61 (m, 1H), 6.19 (m, 1H), 6.51 (dt, 1H), 7.07 (d, 2H),
7.18 (d, 2H); 31P NMR -5.52; HPLC 98%, tR ) 17.8 min, C18,
eluting with a gradient of 0-66% acetonitrile containing 0.1%
TFA, and water containing 0.1% TFA over 22 min; electrospray
mass spectrum (50/50 acetonitrile/water + 0.1% ammonium
hydroxide) m/z calcd 513.5, found 512.6 (M - H) Anal.
(C23H36N3O8P1‚(0.18)H2O‚(0.25)TFA) C, H, N.
4-[(3-Cycloh e xylp r op yl)m e t h ylca r b a m oyl]-4-[3-[6--
(p h osp h on ooxy)n a p h th a len -1-yl]u r eid o]bu tyr ic Acid (7).
(Step 1) To H-Glu(OtBu)-OCH3 (2.0 mmol, 0.50 g) in dichlo-
romethane (25 mL) at 0 °C was added pyridine (8 mmol, 0.66
mL) which was followed by dropwise addition of phosgene in
toluene (3.0 mmol, 1.60 mL, 1.92 M). After the mixture was
stirred at 0 °C for 2 h, 1-amino-6 naphthol (2.0 mmol, 0.32 g)
in dichloromethane was added. After an additional 12 h of
stirring at room temperature, the reaction mixture was
evaporated to dryness and purified on silica gel (1:19, methanol/
dichloromethane) to provide ester 18 as a light brown solid
(0.71 g, 88%): 1H NMR (400 MHz) 1.40 (s, 9H), 1.85 (m, 1H),
2.00 (m, 1H), 2.35 (m, 2H), 3.66 (s, 3H), 4.33 (m, 1H), 7.11 (m,
2H), 7.34 (m, 2H), 7.69 (d, 1H), 7.92 (d, 1H); CI MS (1%NH3
in CH4) m/z calcd 402.3, found 403.4 [M + H]+.
(Step 2) To (6-hydroxynaphthalen-1-yl)ureido-L-Glu(OtBu)-
OCH3 18 (5 mmol, 2.0 g) was added potassium hydroxide (5
mmol, 0.28 g) in water (7 mL). After being heated at 50 °C
for 36 h, the reaction mixture was evaporated to dryness. The
residue was dissolved in water (15 mL), acidified to pH ∼5
using 0.50 N hydrochloric acid, and extracted with the ethyl
acetate (4 × 50 mL) to provide product 19 as a light brown oil
(1.20 g, 62%): 1H NMR (300 MHz) 1.52 (s, 9H), 1.98 (m, 1H),
2.16 (m, 1H), 2.43 (m, 2H), 4.38 (m, 1H), 7.05 (d, 1H), 7.21 (m,
2H), 7.24 (m, 2H), 7.90 (d, 2H), 8.10 (d, 1H), 8.75 (s, 1H);
electrospray mass spectrum (50/50 acetonitrile/water + 0.1%
ammonium hydroxide) m/z calcd 388.3, found 387.5 (M - H).
(Step 3) (6-Hydroxynaphthalen-1-yl)uredio-L-Glu(OtBu)-OH,
19 (0.50 g, 1.3 mmol), was coupled with methyl (3-cyclohexyl-
propyl)amine hydrochloride (0.25 g, 1.3 mmol) using the
procedure previously reported.20 The crude product was
purified on silica gel (2% methanol/CH2Cl2), 20 (0.49 g, 72%):
1H NMR (DMSO/D2O, 400 MHz) 0.80 (m, 2H), 1.17 (m, 7H),
1.40 (s, 9H), 1.62 (m, 7H), 1.90 (m, 1H), 2.30 (m, 2H), 2.82,
3.07 (s, 3H, rotational isomers), 3.10-3.55 (m, 2H), 4.77 (m,
1H), 7.10 (m, 2H), 7.31 (m, 2H), 7.75 (m, 1H), 7.96 (m, 1H).
(Step 4) The naphthol 20 was phosphitylated, oxidized,
deprotected, and purified in a manner similar to that previ-
ously described,20 giving 7 (27 mg): 1H NMR (DMSO/D2O, 400
MHz) 0.82 (m, 2H), 1.17 (m, 6H), 1.48 (m, 1H), 1.52 (m, 7H),
1.90 (m, 1H), 2.35 (m, 2H), 2.84, 3.08 (s, 3H, rotational
isomers), 3.20-3.50 (m, 2H), 4.76 (m, 1H), 7.40 (m, 2H), 7.50
(d, 1H), 7.63 (s, 1H), 7.90 (dd, 1H), 8.10 (dd, 1H); HPLC 100%,
tR 18.9 min, C18, eluting with a gradient of 0-66% acetonitrile
containing 0.1% TFA and water containing 0.1% TFA over 22
min; electrospray mass spectrum (50/50 acetonitrile/water +
0.1% ammonium hydroxide) m/z calcd 549.4, found 548.4 (M
- H) Anal. (C26H36N3O8P1‚(0.3)H2O)‚(0.27)TFA) C, H, N.
(2R )-2-[(t er t -B u t y lo x y c a r b o n y l)m e t h y l]-3-(4-h y -
d r oxyp h en yl)p r op ion ic Acid (8). Intermediate 8 was
synthesized in direct analogy to known procedure:24 1H NMR
(CDCl3, 400 MHz) 1.42 (s, 9H), 2.37 (m, 1H), 2.55 (m, 1H),
2.72 (m, 1H), 3.00 (m, 2H), 6.76 (d, 2H), 7.04 (d, 2H); APCI
MS (1:4, water/acetonitrile) m/z calcd 280.3, found 279.4 [M
- H]. Anal. (C15H20O5) C, H, N.
(Step 2) The crude urea 13 was phosphitylated, oxidized,
deprotected, and purified in a manner similar to that previ-
ously described,20 giving 5 (49 mg) as a colorless solid: 1H NMR
(400 MHz) 0.92 (m, 2H), 1.03-1.25 (m, 6H), 1.42 (m, 1H), 1.62
(m, 7H), 1.78 (m, 1H), 2.24 (t, 2H), 2.79, 3.00 (s, 3H, rotational
isomers), 3.02-3.42 (m, 2H), 3.82 (m, 2H), 4.41 (m, 2H), 4.55
(m, 1H), 6.53 (dd, 1H), 7.10 (d, 2H), 7.23 (d, 2H); 31P NMR
-5.57; HPLC 100%, tR 17.4 min, C18, eluting with a gradient
of 0-66% acetonitrile containing 0.1% TFA and water contain-
ing 0.1% TFA over 22 min. Electrospray mass spectrum (50/
50 acetonitrile/water + 0.1% ammonium hydroxide) m/z calcd
571.5, found 570.3 (M - H). Anal. (C25H38N3O10P1‚(0.13)H2O‚
(0.74)TFA) C, H, N.
(S)-4-[(3-Cycloh exylp r op yl)m eth ylca r ba m oyl]-4-[3-[4-
(p h osp h on ooxy)ben zyl]u r eid o]bu tyr ic Acid (6). (Step 1)
Compound 6 was synthesized in a manner similar to that
described for 5 only 4-benzyloxybenzylamine, 12, was reacted
with the isocyanate to provide 14: 1H NMR (300 MHz) 0.80
(m, 2H), 1.10 (m, 6H), 1.37 (s, 9H), 1.20-1.80 (m, 9H), 1.90
(m, 2H), 2.77, 2.98 (s, 3H, rotational isomers), 3.03-3.40 (m,
2H), 4.10 (d, 2H), 4.60 (m, 1H), 5.05 (s, 2H), 6.14 (dd, 1H),
6.40 (dt, 1H), 6.90-7.42 (m, 9H); electrospray mass spectrum
(50/50 acetonitrile/water + 0.1% ammonium hydroxide) m/z
calcd 579.7, found 580.5 (M - H).
Ack n ow led gm en t. We thank Dr. Gary McClusky
and Colleagues for providing the spectroscopic and
analytical data. We thank Professor Nowick for gener-
ously providing us with his procedure for synthesizing
(Step 2) 14 (520 mg, 0.90 mmol) was dissolved in THF/
methanol (4:1, 50 mL), 5% Pd/C (10%) was added, and the
mixture was place under 1 atm of hydrogen for 12 h. The
reaction was stopped prematurely to avoid possible hydro-

Synthesis of a Nonpeptide Src SH2 Domain Ligand
J ournal of Medicinal Chemistry, 1997, Vol. 40, No. 23 3725
(19) Plummer, M. S.; Lunney, E. A.; Para, K. S.; Prasad, J . V. N. V.;
Shahripour, A.; Singh, J .; Stankovic, C. J .; Humblet, C.; Fergus,
J . H.; Marks, J . S.; Sawyer, T. K. Hydrophobic D-amino acids
ureas prior to publication. Finally, we thank Dr. Charles
J . Stankovic for helpful discussions and assistance in
reviewing the manuscript.
in the design of peptide ligands for the pp60src
SH2 domain. Drug
Des. Disc. 1996, 13, 75-81.
(20) Plummer, M. S.; Lunney, E.; Para, K. S.; Shahripour, A.;
Stankovic, C. J .; Humblet, C.; Fergus, J . H.; Marks, J . S.;
Herrera, R.; Hubbell, S.; Saltiel, A.; Sawyer, T. K. Design of
peptidomimetic ligands for the pp60src SH2 domain. Bioorg.
Med. Chem. 1997, 5, 41-47.
Su p p or tin g In for m a tion Ava ila ble: A description of the
methods used to produce the v-Src protein employed in the
crystallization experiments (3 pages). Ordering information
is given on any current masthead page.
(21) Shahripour, A.; Plummer, M. S.; Lunney, E. A.; Vara Prasad,
J .; Singh, J .; Para, K. S.; Stankovic, C. J .; Eaton, S. R.; Marks,
J . S.; Decker, S. J .; Herrera, R.; Hubbell, S.; Saltiel, A. R.;
Sawyer, T. K. Novel phosphotyrosine and hydrophobic D-amino
acid replacements in the design of peptide ligands for pp60src
SH2 domain. In Peptides: Chemistry, Structure and Biology;
Kaumaya, P. T. P., Hodges, R. S., Eds.; Mayflower Scientific
Ltd.: Kingswinford, England, 1995; pp 394-396.
(22) Shahripour, A.; Plummer, M. S.; Lunney, E.; Para, K. S.;
Stankovic, C. J .; Rubin, J . R.; Humblet, C.; Fergus, J . H.; Marks,
J . S.; Herrera, R.; Hubbell, S. E.; Saltiel, A. R.; Sawyer, T. K.
Novel phosphotyrosine mimetics in the design of peptide ligands
for pp60src SH2 domain. Bioorg. Med. Chem. Lett. 1996, 6,
1209-1214.
(23) Fields, G. B.; Noble, R. L. Solid-phase peptide synthesis utilizing
9-fluorenylmethoxycarbonyl amino acids. Int. J . Pept. Protein
Res. 1990, 35, 161-214.
(24) Plattner, J . J .; Marcotte, P. A.; Kleinert, H. D.; Stein, H. H.;
Greer, J .; Bolis, G.; Fung, A. K. L.; Bopp, B. A.; Luly, J . R.; Sham,
H. L.; Kempf, D. J .; Rosenberg, S. H.; Dellaria, J . F.; De, B.;
Merits, I.; Perun, T. J . Renin inhibitors. Dipeptide analogs of
angiotensinogen utilizing a structurally modified phenylalanine
residue to impart proteolytic stability. J . Med. Chem. 1988, 31,
2277-2288.
(25) Nowick, J . S.; Holmes, D. L.; Noronha, G.; Smith, E. M.; Nguyen,
T. M.; Huang, S.-L. Synthesis of peptide isocyanates and
isothiocyanates. J . Org. Chem. 1996, 61, 3929-3934.
(26) Stankovic, C. J .; Surendran, N.; Lunney, E.; Plummer, M. S.;
Para, K. S.; Shahripour, A.; Humblet, C.; Fergus, J . H.; Marks,
J . S.; Herrera, R.; Hubbell, S. E.; Saltiel, A. R.; Stewart, B. H.;
Sawyer, T. K. The Role of 4-Phosphonodifluoromethyl- and
4-phosphono-phenylalanine in the selectivity and cellular uptake
of SH2 domain ligands. Bioorg. Med. Chem. Lett. 1997, 7 (14),
1909-1914.
(27) Smyth, M. S.; Ford, H., J r.; Burke, T. R., J r. A general method
for the preparation of benzylic R,R-difluorophosphonic acids; non-
hydolyzable mimetics of phosphotyrosine. Tetrahedron Lett.
1992, 33, 4137-4140.
(28) Burke, T. R., J r.; Smyth, M. S.; Otaka, A.; Nomizu, M.; Roller,
P. P.; Wolf, G. F.; Case, R.; Shoelson, S. E. Nonhydrolyzable
phosphotyrosyl mimetics for the preparation of phosphatase-
resistant SH2 domain inhibitors. Biochemistry 1994, 33, 6490-
6494.
(29) Details of the protein preparation used in the crystallography
experiments is available as Supporting Information (3 pages).
Ordering information is given on any current masthead page.
(30) Zuckermann, R. N.; Martin, E. J .; Spellmeyer, D. C.; Stauber,
G. B.; Shoemaker, K. R.; Kerr, J . M.; Figliozzi, G. M.; Goff, D.
A.; Siani, M. A.; Simon, R. J .; Banville, S. C.; Brown, E. G.;
Wang, L.; Richter, L. S.; Moos, W. H. Discovery of nanomolar
ligands for 7-transmembrane G-protein-coupled receptors from
a diverse N-(substituted)glycine peptoid library. J . Med. Chem.
1994, 37, 2678-2685.
Refer en ces
(1) Koch, C. A.; Anderson, D.; Moran, M. F.; Ellis, C.; Pawson, T.
SH2 and SH3 domains: elements that control interactions of
cytoplasmic signaling proteins. Science 1991, 252, 668-674.
(2) Cantley, L. C.; Auger, K. R.; Carpenter, C.; Duckworth, B.;
Graziani, A.; Kapeller, R.; Soltoff, S. Oncogenes and signal
transduction. Cell 1991, 64, 281-302.
(3) Pawson, T.; Gish, G. D. SH2 and SH3 domains: from structure
to function. Cell 1992, 71, 359-362.
(4) Alonso, G.; Koegl, M.; Mazurenko, N.; Courtneidge, S. A.
Sequence requirements for binding of Src family tyrosine kinases
to activated growth factor receptors. J . Biol. Chem. 1995, 270,
9840-9848.
(5) Luttrell, D. K.; Lee, A.; Lansing, T. J .; Crosby, R. M.; J ung, K.
D.; Willard, D.; Luther, M.; Rodriguez, M.; Berman, J .; Gilmer,
T. M. Involvement of pp60src with two major signaling pathways
in human breast cancer. Proc. Natl. Acad. Sci. U.S.A. 1994, 91,
83-87.
(6) Roussel, R. R.; Brodeur, S. R.; Shalloway, D.; Laudano, A. P.
Selective binding of activated pp60c-src by an immobilized
synthetic phosphopeptide modeled on the carboxyl terminus of
pp60c-src. Proc. Natl. Acad. Sci. U.S.A. 1991, 88, 10696-10700.
(7) Payne, G.; Shoelson, S. E.; Gish, G. D.; Pawson, T.; Walsh, C.
T. Kinetics of p56lck and p60src Src homology 2 domain binding
to tyrosine-phosphorylated peptides determined by a competition
assay or surface plasmon resonance. Proc. Natl. Acad. Sci. U.S.A.
1993, 90, 4902-4906.
(8) Payne, G.; Stolz, L. A.; Pei, D.; Band, H.; Shoelson, S. E.; Walsh,
C. T. The phosphopeptide-binding specificity of Src family SH2
domains. Chem. Biol. 1994, 1, 99-105.
(9) Boyce, B. F.; Yoneda, T.; Lowe, C.; Soriano, P.; Mundy, G. R.
Requirement of pp60c-src expression for osteoclasts to form
ruffled borders and resorb bone in mice. J . Clin. Invest. 1992,
90 (4), 1622-1627.
(10) Soriano, P.; Montgomery, C.; Geske, R.; Bradley, A. Targeted
disruption of the c-src proto-oncogene leads to osteopetrosis in
mice. Cell 1991, 64, 693-702.
(11) Songyang, Z.; Shoelson, S. E.; Chaudhuri, M.; Gish, G.; Pawson,
T.; Haser, W. G.; King, F.; Roberts, T.; Ratnofsky, S.; Lechleider,
R. J .; Neel, B. G.; Birge, R. B.; Fajardo, J . E.; Chou, M. M.;
Hanafusa, H.; Schaffhausen, B.; Cantley, L. C. SH2 domains
recognize specific phosphopeptide sequences. Cell 1993, 72, 767-
778.
(12) Songyang, Z.; Shoelson, S. E.; McGlade, J .; Olivier, P.; Pawson,
T.; Bustelo, X. R.; Barbacid, M.; Sabe, H.; Hanafusa, H.; Yi, T.;
Ren, R.; Baltimore, D.; Ratnofsky, S.; Feldman, R. A.; Cantley,
L. C. Specific motifs recognized by the SH2 domains of Csk,
3BP2, fps/fes, GRB-2, HCP, SHC, Syk, and Vav. Mol. Cell. Biol.
1994, 14, 2777-2785.
(13) Waksman, G.; Kominos, D.; Robertson, S. C.; Pant, N.; Balti-
more, D.; Birge, R. B.; Cowburn, D.; Hanafusa, H.; Mayer, B.
J .; Overduin, M.; Resh, M. D.; Rios, C. B.; Silverman, L.;
Kuriyan, J . Crystal structure of the phosphotyrosine recognition
domain SH2 of v-src complexed with tyrosine-phosphorylated
peptides. Nature 1992, 358, 646-653.
(14) Waksman, G.; Shoelson, S. E.; Pant, N.; Cowburn, D.; Kuriyan,
J . Binding of a high affinity phosphotyrosyl peptide in the Src
SH2 domain: Crystal structures of the complexed and peptide-
free forms. Cell 1993, 72, 779-790.
(15) Xu, W.; Harrison, S. C.; Eck, M. J . Three-dimensional structure
of the tyrosine kinase c-Src. Nature 1997, 385, 595-602.
(16) Stankovic, C. J .; Plummer, M. S.; Sawyer, T. K. Peptidomimetic
ligands for Src homology-2 domains. In Advances in amino acid
mimetics and peptidomimetics; Abell, A., Ed.; J AI Press: Green-
wich, CT; Vol. 1, pp 127-163 (in press).
(17) Gilmer, T.; Rodriguez, M.; J ordan, S.; Crosby, R.; Alligood, K.;
Green, M.; Kimery, M.; Wagner, C.; Kinder, D.; Charifson, P.;
Hassel, A. M.; Willard, D.; Luther, M.; Rusnak, D.; Sternbach,
D. D.; Mehrotra, M.; Peel, M.; Shampine, L.; Davis, R.; Robins,
J .; Patel, I. R.; Kassel, D.; Burkhart, W.; Moyer, M.; Bradshaw,
T.; Berman, J . Peptide inhibitors of src SH3-SH2-phospho-
protein interactions. J . Biol. Chem. 1994, 269, 31711-31719.
(18) Rodriguez, M.; Crosby, R.; Alligood, K.; Gilmer, T.; Berman, J .
Tripeptides as selective inhibitors of src-SH2 phosphoprotein
interactions. Lett. Pept. Sci. 1995, 2, 1-6.
(31) Zuckermann, R. N.; Kerr, J . M.; Kent, S. B. H.; Moos, W. H.
Efficient method for the preparation of peptoids [oligo(N-
substituted glycines)] by submonomer solid-phase synthesis. J .
Am. Chem. Soc. 1992, 114, 10646-10647.
(32) Holland, D. R., personal communication.
(33) Kabsch, W. Recent extensions of the data processing program
XDS; Proceedings of the CCP4 study weekend; Sawyer, L.,
Isaacs, N., Bailey, S., Eds.; SERC Daresbury Laboratory: War-
rington, U.K., 1993; pp 63-70.
(34) Navaza, J . AmoRe: an automated package for molecular re-
placement. Acta Crystallogr. 1994, A50, 157-163.
(35) Brunger, A. T. X-PLOR Version 3.1; Yale University Press: New
Haven, 1992.
(36) QUANTA Users Guide; v. 4.0 ed.; Molecular Simulations: San
Diego, 1996.
(37) McNamara, D. J .; Dobrusin, E. M.; Zhu, G.; Decker, S. J .; Saltiel,
A. R. Inhibition of binding of phospholipase Cg1 SH2 domains
to phosphorylated epidermal growth factor receptor by phos-
phorylated peptides. Int. J . Pept. Protein Res. 1993, 42, 240-248.
(38) Wallace, A. C.; Laskowski, R. A.; Thorton, J . M. Protein Eng.
1995, 8 (2), 127-134.
J M970402Q